The Journal of Antibiotics (2016) 69, 689–696 & 2016 Japan Antibiotics Research Association All rights reserved 0021-8820/16 www.nature.com/ja

ORIGINAL ARTICLE

Antimicrobial potency of single and combined mupirocin and , thymol, and 1,8-cineole against planktonic and biofilm growth

Domagoj Kifer, Vedran Mužinić and Maja Šegvić Klarić

Staphylococcus aureus is one of the most commonly isolated microbes in chronic rhinosinusitis (CRS) that can be complicated due to the formation of a staphylococcal biofilm. In this study, we investigated antimicrobial efficacy of single mupirocin and three types of monoterpenes (thymol, menthol and 1,8-cineole) as well as mupirocin– combinations against S. aureus ATCC 29213 and 5 methicilin-resistant S. aureus strains (MRSA) grown in planktonic and biofilm form. MIC against planktonic bacteria as well as minimum biofilm-eliminating concentrations (MBECs) and minimum biofilm inhibitory concentrations (MBICs) were determined by TTC and MTT reduction assay, respectively. The MICs of mupirocin (0.125–0.156 μgml− 1) were three orders of magnitude lower than the MICs of monoterpenes, which were as follows: thymol (0.250–0.375 mg ml − 1) 4 menthol (1 mg ml − 1) 4 1,8-cineole (4–8mgml− 1). Mupirocin-monoterpene combinations showed indifferent effect as compared with MICs of single substances. Mupirocin (0.016–2mgml− 1) failed to destroy the biofilm. The MBECs of thymol and menthol were two- to sixfold higher than their MICs, while 1,8-cineole exerted a weak antibiofilm effect with MBECs 16- to 64-fold higher than MICs. Mixture of mupirocin and 1,8 cineole exerted a potentiated biofilm-eliminating effect, mupirocin–menthol showed antagonism, while effect of thymol–mupirocin mixture was inconclusive. MBICs of antimicrobials were close to their MICs, except 1,8-cineole, MBIC was about three- to fivefold higher. Dominant synergy was observed for mixtures of mupirocin and menthol or thymol, whereas mupirocin-1,8-cineol exerted an indifferent or additive biofilm inhibitory effect. Particular combinations of mupirocin and the monoterpenes could be applied in CRS therapy in order to eliminate or prevent bacterial biofilm growth. The Journal of Antibiotics (2016) 69, 689–696; doi:10.1038/ja.2016.10; published online 17 February 2016

INTRODUCTION treatment of S. aureus-associated . It is a structural Staphylococcus aureus is one of the most important human colonizers analog of isoleucyl-adenylate (Ile-AMP), which competes with and pathogens that causes a variety of diseases, from mild skin and Ile-AMP for binding sites of the isoleucyl-tRNA synthetases leading soft tissue infections to more serious conditions including pneumonia, to abrogation of protein synthesis in bacteria.9,10 Recently, Bode endocarditis, osteomyelitis and sepsis.1 Also, it is one of the most et al.11 showed that the nasal decolonization of S. aureus carriers by commonly isolated microbes in chronic rhinosinusitis (CRS), a mupirocin significantly reduces the risk of S. aureus infections after chronic disease of the paranasal sinuses that affects a large number surgery. However, data on the efficacy of mupirocin treatment in of individuals in the developed world.2–4 Apart from the methicilin biofilm-associated nasal S. aureus infections as well as mupirocin resistance of S. aureus (MRSA), infections with S. aureus can even antibiofilm activity in vitro are scarce. The development of antibiotic further complicate owing to biofilm formation. In CRS, mucosal resistance in pathogenic bacteria increased general interest in studying changes result in favorable conditions for the development of a the antimicrobial potency of including monoterpenes S. aureus biofilm. Once a biofilm is established, its resistance to both menthol, thymol and 1,8-cineole. These amphipathic phytochemicals host defences and antimicrobials (restricted penetration, decrease in with prevalent hydrophobicity evoke alterations of S. aureus bacterial metabolism and growth rate, increase in antibiotic-degrading membrane permeability causing leakage of intracellular material.12 enzymes accumulation and enhancement of exchanging rate of genes The studies undertaken so far have shown that some phytochemicals encoding for resistance) leads to chronic inflammation.5–8 Mupirocin including thymol might have impact on bacterial biofilm formation or pseudomonic acid A is natural antibiotic produced by Pseudomonas and development, acting as control agents of bacterial adhesion and fluorescens, which has been successfully used as topical agent in the cell-to-cell communication known as quorum sensing-QS.12 However,

Department of Microbiology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia Correspondence: Professor MŠ Klarić, Department of Microbiology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Schrottova 39, 10000 Zagreb, Croatia. E-mail: [email protected] Received 17 October 2015; revised 16 January 2016; accepted 22 January 2016; published online 17 February 2016 Mupirocin and monoterpenes against Staphylococci DKiferet al 690

the biofilm eradication ability of such phytochemicals is insufficiently Biofilm formation investigated. Taking into account the amphipathic nature of menthol, Biofilm was formed according to the method by Walencka et al.15 with thymol and 1,8-cineole, these monoterpenes could penetrate through minor modifications. Stock inoculum was prepared as described previously − the exopolysaccharide matrix of S. aureus biofilm and enhance the andadjustedto1,5×106 c.f.u. ml 1 in Tryptic soy broth (TSB, Difco, Le Pont activity of mupirocin trough membrane permeability disturbances. de Claix, France), supplemented with 0,25% D-(+)- (Kemika, Zagreb, Therefore, the aim of our study was to investigate the antimicrobial Croatia; TSBGlc). Diluted bacterial suspensions were incubated for 24 h at 37 ° C, aerobically. efficacy of mupirocin alone and the mentioned monoterpenes as well – Following incubation, the suspension was diluted 20-fold with TSBGlc. as mupirocin monoterpene combinations against S. aureus planktonic μ fi A total of 100 l of this suspension was added to each well of 96-well tissue and bio lm modes of growth in vitro. culture plate (Nuncoln Surface, Nunc, Thermo Fisher Scientific, Waltham, MA, USA) and incubated for 24 h at 37 °C aerobically, to form the biofilm. To MATERIALS AND METHODS visualize a biofilm, a method by Kairo et al.16 was used, with a few Bacterial strains modifications. After biofilm formation, wells were emptied and gently rinsed Staphylococcus aureus ATCC 29213 and clinical MRSA isolates (n = 5) were with 100 μl of saline, to remove planktonic bacteria and preserve the biofilm. used in this study. All of the strains (MFBF 10674, MFBF 10676, MFBF 10677, After rinsing, 50 μl of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo- MFBF 10679, MFBF 10680) were taken from the microbe culture collection lium , MTT reagent, Sigma) was added into each well and incubated of Department of Microbiology, Faculty of Pharmacy and Biochemistry, for 2 h at 37 °C. MTT is metabolized by living bacterial cells forming purple University of Zagreb. formazan crystals. The formazan crystals were dissolved by adding 150 μlof dimethyl sulfoxide (DMSO, Sigma). The plate was shaken for 10 min until all Antimicrobials formazan crystals dissolved. The absorbance was measured on a microplate Mupirocin dihydrate was donated by Pliva (Pliva, Zagreb, Croatia), reader (iEMS, Labsystems) at wavelength of 540 nm. whereas monoterpenes L-menthol(Sigma,StLouis,MO,USA),thymol(Kemika, Zagreb, Croatia) and 1,8-cineole (Merck, Darmstadt, Germany) were purchased. Determination of minimum biofilm-eliminating concentration Immediately before the assay, mupirocin calcium dihydrate was dissolved The biofilm of each S. aureus strain was grown on the bottom of wells of a and diluted in an appropriate broth. Monoterpenes were dissolved in 100% 96-well microtiter plate as described above. Initial biofilm was stained and its ethanol (Kemika, Zagreb, Croatia) and diluted in an appropriate broth. Stock absorbance was measured as described above. The obtained absorbance was solution of Tween 80 in sterile water (5%) was used for homogenization of defined as the absorbance of the initial biofilm, Aib. monoterpene alcoholic solutions. In other wells, the intact initial biofilm was treated by adding 100 μlof Determination of MIC against planktonic bacteria antimicrobial agent solution. Concentration ranges of antimicrobials − − − A stock culture of S. aureus strains was prepared, grown at 37 °C aerobically for were 0.016 μgml 1–2mgml 1 (mupirocin), 0.5–64 mg ml 1 (menthol), − − 24 h in Müller–Hinton broth (Merck, Germany). A 1.5 × 108 colony-forming 0.25–32 mg ml 1 (thymol) and 2–256 mg ml 1 (1,8-cineole). Concentrations unit (c.f.u.) per ml inoculum was prepared using a nephelometer (BioMereiux, were prepared by diluting stock solutions with Tryptic soy broth (TSB, Merck, Marcy-l'Etoile, France) and adjusted to a final concentration 5 × 105 c.f.u. ml − 1 Germany). Control strains were grown in broth with and without 5–10% of in Müller–Hinton broth containing antimicrobials. The determination of MICs ethanol + tween. Plates were incubated for 24 h at 37 °C aerobically. Following for mupirocin, menthol, thymol and 1,8-cineole were carried out using a incubation, the wells were emptied and rinsed, and the biofilm remaining on twofold microdilution method, according to Clinical and Laboratory Standards the bottom of the wells was stained and measured as described previously. The 13 Institute guidelines. Concentrations of twofold serial-diluted solutions in obtained absorbance was defined as the absorbance of surviving biofilm, Asb. Müller–Hinton broth used for determining MIC for mupirocin were in a Antimicrobial treatment of each strain as well as controls was derived in − 1 range from 0.0625 to 128 μgml , concentrations of menthol, thymol and quadruplicate. Viability was calculated using Aib and Asb by Equation (2). 1,8-cineole were in ranges of 0.03125–64 mg ml − 1,0.125–128 mg ml − 1 and (Minimum biofilm-eliminating concentration (MBEC) is defined as minimum 0.125–128 mg ml − 1, respectively. Also, a determination of MIC for concentration of antimicrobial agent solution in well whose viability is ⩽ 10%). combinations of mupirocin and each of monoterpenes was carried out. Concentrations of twofold serial-diluted solutions used for assessing MIC ¼ Asb ´ ð Þ – μ − 1 Viability 100 2 of combinations were in ranges of 0.015625 0.5 gml (mupirocin), Aib 0.5–4mgml− 1 (menthol), 0.125–8mgml− 1 (thymol) and 0.125–32 mg ml − 1 According to Chou,17 results were plotted into corresponding linear form of (1,8-cineole). Concentration gradients were decreased in the same direction À median-effect plot (y ¼ log ð1 viabilityÞ, x = log (concentration)) and using and the MICs of both compounds were in the same well. Culture of S. aureus in 2 viability 2 the broth with 0.31–1.25% of ethanol and 0.016–0.063% tween was used as linear regression, MBEC was determined as a concentration where viability positive (growth) control, whereas broth without S. aureus as negative control. was 10% (Equation (3)). Plates were incubated for 24 h, at 37 °C in aerobic atmosphere. At the end of À : log ðÞ1 0 1 Àb incubation, 2,3,5-triphenyltetrazolium chloride (TTC, Sigma) was added as a 2 0:1 MBE concentration ¼ 2 a ð3Þ visualization agent to the final concentration of 0.5 mg ml − 1. Surviving bacteria metabolize the TTC and produce red formazan. MIC was derived from a Mupirocin was combined with each monoterpene separately. The method is microtitre plate as the minimum concentration of antimicrobial agent in the similar to the determination of MIC for combinations—the concentration unstained wells. Examination for every strain was derived in quadruplicate. gradient went in the same direction. A total of 50 μl of mupirocin solution was Mean concentration was used as a final result. added into each well, and 50 μl of monoterpene solution to final concentration To simplify interpretation of MIC for combinations, Fractional ranges of 0.016–0.125 μgml− 1 (mupirocin), 0.5–4mgml− 1 (menthol), Inhibitory Concentration Index (FICI) was calculated using an equation 0.125–1mgml− 1 (thymol) and 16–128 mg ml − 1 (1,8-cineole). Concentrations (Equation (1)). The combination of two antimicrobial agents is consider to were prepared by diluting stock solutions with TSB. Plates were incubated for 4 be synergistic when FICI is less or equal to 0.5, additive when it is 0.5 and 24 h at 37 °C in aerobic atmosphere. Controls were grown in the same manner ⩽ 4 ⩾ 4 1, indifferent when it is 1and 4.0, and antagonistic when it is 4(cited as described previously. After incubation, the biofilm remained on the bottom in ref. 14). of the wells was visualized as described earlier. The obtained absorbance was fi fi Xn de ned as absorbance of survived bio lm (Asb). Using absorbances of initial MICðÞ animicrobial agent in combination FICI ¼ ð1Þ and survived biofilm, viability and MBEC were calculated by the above- MICðÞ antimicrobial agent alone i¼1 mentioned equations (Equations (2) and (3)). All experiments were performed in quadruplicate.

The Journal of Antibiotics Mupirocin and monoterpenes against Staphylococci DKiferet al 691

Table 1 Antimicrobial activity of single mupirocin and monoterpenes against planktonic Staphylococci (MICs) and their biofilm (MBECs and MBICs)

Mupirocin Thymol Menthol 1,8-cineole

MIC MBIC MIC MBEC MBIC MIC MBEC MBIC MIC MBEC MBIC S. aureus strain (μgml− 1) (μgml− 1) (mg ml − 1) (mg ml − 1) (mg ml − 1) (mg ml − 1) (mg ml − 1) (mg ml − 1) (mg ml − 1) (mg ml − 1) (mg ml − 1)

ATCC 29213 0.125 0.411 0.25 1.07 0.43 1 3.21 0.64 8 128 16.75 MFBF 10674 0.125 0.142 0.25 1.05 0.33 1 3.24 1.98 8 254 19.12 MFBF 10676 0.125 0.366 0.25 0.76 0.41 1 6.35 1.58 8 246 38.14 MFBF 10677 0.156 0.158 0.25 0.93 0.40 1 3.28 0.88 4 254 14.20 MRSA 10679 0.125 0.332 0.375 0.59 0.59 1 4.19 1.67 8 245 20.95 MRSA 10680 0.125 0.114 0.25 0.53 0.43 1 3.73 1.33 8 237 30.31

Abbreviations: MBIC, minimum biofilm inhibitory concentration; MBEC, minimum biofilm-eliminating concentration; MRSA, methicilin-resistant S. aureus strains. MFBF 10674–10680 refers to S. aureus MRSA strains from microbe culture collection of Department of Microbiology, Faculty of Pharmacy and Biochemistry, University of Zagreb. Linear regression coefficintes (R2) for MBEC were in range from 0.831 to 1 and for MBIC were from 0.819 to 0.997.

Determination of minimum biofilm inhibitory concentration Among the monoterpenes, thymol showed the most potent Bacterial inoculums were prepared and adjusted to a concentration of 1.5 × 106 antimicrobial activity (0.250–0.375 mg ml − 1), followed by menthol − c.f.u. ml 1 in TSBGlc as described previously. Upon 24 h of incubation at (1 mg ml − 1) and 1,8-cineole (4–8mgml− 1). According to the 37 °C, bacterial suspensions were 10-fold diluted in TSBGlc. calculated FIC Index (Table 2), all of the tested combinations showed Atotalof50μl of antimicrobial agent was added to the wells of 96-well indifferent effect except for mupirocin and thymol combination tested plates in final concentration ranges of 0.016–1 μgml− 1 (mupirocin), 0.5–16 mg ml − 1 (menthol), 0.063–4mgml− 1 (thymol) and 4–128 mg ml − 1 against MRSA strains MFBF 10674, 10677 and 10680 where an (1,8-cineole). The concentrations were prepared by diluting stock solutions additive effect was observed. with TSBGlc. Controls were treated with 0.63–5% of ethanol in TSBGlc. After achieving a concentration gradient, 50 μl of prepared bacterial suspensions were Biofilm-eliminating effect added to each well. The plates were incubated aerobically for 24 h at 37 °C. All S. aureus strains produced a solid biofilm on polystyrene Upon incubation, biofilm was visualized as described previously. microplates. Biofilms of all tested S. aureus strains showed resistance Viability was calculated using the absorbance of biofilm formed by untreated to mupirocin in all tested concentrations (0.016 μgml− 1–2mgml− 1). fi bacteria (Aut) and absorbances of bio lm in treated bacteria (At) according to Biofilm viability varied from 41.35 to 103.78% without any pattern Equation (4). through an increasing concentration of mupirocin (Figure 1). On the fi At other hand, monoterpenes reduced bio lm viability down to 10%. Viability ¼ ´ 100 ð4Þ fi Aut Similar to MIC values, antibio lm effect was best shown by – − 1 MBIC is defined similarly as MBEC—using linear regression after plotting thymol (BEC 0.59 1.07 mg ml ), followed by menthol (BEC – − 1 4 − 1 results into corresponding linear form of median-effect plot and calculating the 3.21 6.35 mg ml ) and 1,8-cineole (BEC 128 mg ml ) (Table 1). concentration needed for viability of 10% (Equation (3)). To test the biofilm eliminaton ability of mupirocin–monoterpene The MBIC of mupirocin − monoterpene combinations were determined as combinations, mupirocin was applied in the range of concentrations follows. A total of 25 μl of mupirocin solution was added to the wells to set a (0.016–0.125 μgml− 1) that reduced biofilm viability by approximately − 1 final concentration range of 0.002–0.125 μgml in combination with thymol 10–60%, whereas monoterpenes were applied at concentration fi – − 1 and 1,8-cineole, and nal concentration range of 0.0005 0.016 mg ml in ranges that cover MBEC. The biofilm-eliminating effects of selected combination with menthol. Afterwards, 25 μl of monoterpene solution was − mupirocin–monoterpene combinations in comparison with mono- added to wells in final concentration ranges of 0.125–1mgml 1 (menthol), 0.016–1mgml− 1 (thymol) and 1–64 mg ml − 1 (1,8-cineole). The concentra- applied alone are presented in Figures 2, 3 and 4. Combina- − 1 μ − 1 tions were prepared by diluting stock solutions with TSBGlc. A total of 50 μlof tion of menthol (4 mg ml ) and mupirocin (0.125 gml ) showed bacterial suspension 10 times diluted in TSBGlc was added into each well. The antagonism; single menthol-reduced biofilm by 90%, whereas com- plate was incubated for 24 h at 37 °C and viable biofilm was measured as bined treatment eliminated between 40 and 80% of biofilm. Opposite described above. All experiments were performed in quadruplicate. Interactions to that, thymol (0.5 mg ml − 1) and mupirocin (0.063 μgml− 1)exerted fi – were de ned by FIC Index (Equation (1)), in terms of MBIC of mupirocin potentiated effect, but only to three bacterial strains (Po0.05), monoterpene combination and MBIC of antimicrobial agent applied alone. whereas 1,8-cineol (128 mg ml − 1) in combination with mupirocin (0.125 μgml− 1) showed significant potentiated biofilm-eliminating Statistics effect against all tested strains (Po0.05) and biofilm viability dropped Statistically significant differences between staphylococcal viability observed upon treatment with single monoterpene and monoterpene-mupirocin combi- to ~ 10%. nation were analyzed by unpaired t-test. The level of Po0.05 was considered fi statistically significant. Bio lm inhibitory effect The MBICs of mupirocin and monoterpenes applied alone that RESULTS inhibited biofilm formation of each S. aureus strain by 90% are Antimicrobial activity against planktonic Staphylococci presented in Table 1. Mupirocin inhibited biofilm formation in − MICs for mupirocin, menthol, thymol and 1,8-cineole against tested concentrations close to its MICs (0.114–0.411 μgml 1). MBICs of − − bacterial strains are presented in Table 1. Mupirocin showed the menthol (0.64–1.98 mg ml 1) and thymol (0.33–0.59 mg ml 1)were strongest antimicrobial activity; MICs (0.125–0.156 μgml− 1) were also closely to their MIC values, while MBICs of 1,8-cineole were three orders of magnitude lower than the MICs of monoterpenes. approximately three- to fivefold higher than MICs.

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Applying mupirocin–monoterpene combinations and calculating S. aureus strain ATCC 29213 as well as against MRSA strains. The FIC Index, a dominant synergy was observed for mixtures of bacterial strains did not show significant difference in susceptibility mupirocin and menthol or thymol, whereas the muprirocin and with respect to a single antimicrobial agent. Among the tested 1,8-cineol combination exerted an indifferent or additive biofilm antimicrobials, mupirocin exerted the most potent activity and 18–20 inhibitory effect (Table 3). MIC values were in accordance with previous reports. The antimicrobial potency of monoterpenes against S. aureus was as follows; MICs thymoloMICs mentholoMICs 1,8-cineole, and DISCUSSION corresponded to literature data.21–24 Trombeta et al.22 proposed a The standard assay for testing the antimicrobial susceptibility of mechanism of antibacterial action for phenolic terpene thymol and planktonic bacteria demonstrated that mupirocin and monoterpenes alcoholic terpene menthol. These lipophilic compounds possess menthol, thymol and 1,8-cineole applied alone were effective against detectable water and could migrate across the aqueous extracellular medium and interact with membrane lipids resulting in Table 2 Antimicrobial efficacy of mupirocin–monoterpene alterations of membrane permeability and leakage of intracellular combinations against planktonic S. aureus materials. In addition, monoterpene transfer into the bacteria and interaction with intracellular structures could not be excluded.22 MICmupirocin MICmonoterpene Taking into account the structure of 1,8-cineole we can assume that S.aureus strain (μgml− 1) (mg ml − 1)FICIEffect this cyclic ether monoterpene might have a similar mechanism of antibacterial action as was proposed for thymol and menthol. On the Thymol other hand, mupirocin is a hydrophilic antibiotic that enters into a ATCC 29213 0.125 0.25 2.0 Indifferent bacterial cell by passive diffusion and interferes in protein synthesis by MFBF 10674 0.063 0.125 1.0 Additive blocking the enzyme isoleucyl-tRNA synthetase.9,10,25 On the basis of MFBF 10676 0.125 0.25 2.0 Indifferent the proposed antibacterial mechanisms we hypothesized that these MFBF 10677 0.063 0.125 0.9 Additive compounds may act synergistically or at least additively. Surprisingly, MFBF 10679 0.125 0.25 1.7 Indifferent mupirocin–monoterpene combinations showed a dominant MFBF 10680 0.063 0.125 1.0 Additive indifferent effect against planktonic S. aureus strains. fi Menthol Before we continue discussing the results on S. aureus bio lm ATCC 29213 0.125 0.25 2.0 Indifferent susceptibility to mupirocin and monoterpenes, we would like to fi MFBF 10674 0.063 0.125 2.0 Indifferent address the methodology for assessing the antibio lm effects reported MFBF 10676 0.125 0.25 2.0 Indifferent in the literature. Several colorimetric methods for biofilm susceptibility MFBF 10677 0.063 0.125 1.8 Indifferent testing have been described for measuring active bacterial metabolism 15,26–28 29,30 MFBF 10679 0.125 0.25 2.0 Indifferent in biofilm, or biofilm staining protocols as well as turbidity MFBF 10680 0.063 0.125 2.0 Indifferent measuring upon biofilm growth using Calgary Biofilm Device.31 Thus, a single standard method for biofilm susceptibility testing still does not 1.8-cineole exist as well as a univocal interpretation of results. This makes the ATCC 29213 0.125 8 2.0 Indifferent comparison with already published results very difficult. In this study, MFBF 10674 0.125 8 2.0 Indifferent the concentration that decreased bacterial viability, determined as MFBF 10676 0.125 8 2.0 Indifferent metabolic activity in biofilm, to ⩽ 10% was defined as minimum MFBF 10677 0.0625 4 1.4 Indifferent biofilm-eliminating concentration (MBEC). Keeping in mind that MFBF 10679 0.125 8 2.0 Indifferent antimicrobials affect mature biofilm, we may suggest that it is more MFBF 10680 0.125 8 2.0 Indifferent appropriate to use the term MBEC rather than MBIC, which has Abbreviation: FICI, Fractional Inhibitory Concentration Index. been applied in some reports.15,32 On the other hand, the term

Mupirocin 120

100

80

60

Viability (%) 40

20

0 0.015625 0.03125 0.0625 0.125 0.25 0.5 1 2 Mupirocin (mg ml-1)

ATCC 29213 MFBF 10674 MFBF 10767 MFBF 10677 MFBF 10679 MFBF 10680

Figure 1 Biofilm-eliminating effect of single mupirocin on Staphylococcus aureus strains.

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Thymol 50 45 40 35 30 * 25 * 20 Viability (%) * 15 * * 10 5 0 ATCC 29213 MFBF 10674 MFBF 10676 MFBF 10677 MFBF 10679 MFBF 10680 S. aureus strains

Thymol (0.5 mg mL-1) Thymol (0.5 mg mL-1) + mupirocin (0.063 µg mL-1)

Figure 2 Biofilm-eliminating efficacy of single thymol in comparison with thymol–mupirocin combination. Data are represented as viability mean ± s.d. * represent statistically significant difference (Po0.05).

Menthol 70 * * 60 * 50

40 * * * 30 Viability (%) 20

10

0 ATCC 29213 MFBF 10674 MFBF 10676 MFBF 10677 MFBF 10679 MFBF 10680 S. aureus strains Menthol (4 mg mL-1) Menthol (4 mg mL-1) + mupirocin (0.125 µg mL-1)

Figure 3 Biofilm-eliminating efficacy of single menthol in comparison with menthol–mupirocin combination. Data are represented as viability mean ± s.d. * represent statistically significant difference (Po0.05).

MBIC is more convenient to indicate the minimal concentration 50% even with the highest concentration of antibiotic (0.2 mg ml − 1). of an antimicrobial agent that inhibits the formation of biofilm. This could be due to the overproduction of exopolysaccharide which Such terminology has been used in several reports,26–28,33 including protects metabolically active bacteria embedded in the biofilm com- the present study. munity and mupirocin could eliminate only the cells closest to the It is well known that bacteria in biofilm undergo phenotypic liquid-biofilm interface. Another study showed that mupirocin applied changes that make them several-fold more resistant to antimicrobials; in low concentrations (7.81–125 μgml− 1) reduced 8-day-old S. aureus for example, exopolysaccharide matrix may prevent the access of biofilm grown in cerebrospinal fluid broth by 90%.32 As was antibiotics to the bacteria, or cells within biofilm could have different mentioned above, comparison is very difficult in this case owing to growth characteristics and take up nutrients and drugs differently from different methodologies. planktonic bacteria.34,35 Contrary to strong effect against planktonic Unlike mupirocin, thymol and menthol were able to reduce biofilm S. aureus, mupirocin failed in destroying bacterial biofilm as by 90% at concentrations two- to sixfold higher than their MICs. determined by biofilm-eliminating MTT reduction assay. Low con- 1,8-cineole exerted a weak antibiofilm effect to all S. aureus strains centrations of mupirocin (0.016–0.125 μgml− 1) reduced biofilm by with MBECs 16- to 64-fold higher than MICs. Contrary to biofilm 10–60%, whereas an increase in concentration of up to 2 mg ml − 1 did eradication potency, all of the tested antimicrobials inhibited biofilm not have a significantly greater antibiofilm effect. Similar results were formation in concentrations close to their MICs, except 1,8-cineole obtained by Hurler et al.26 who determined antibiofilm effects of whose MBIC was about three- to fivefold higher. The biofilm mupirocin using resazurin reduction assay. When mupirocin was inhibitory properties of thymol are well documented, but the applied at a concentration of 0.5 μgml− 1,40%ofS. aureus biofilm biofilm eradication potencies of all three monoterpenes are poorly reduction was observed, but biofilm eradication rate did not exceed investigated. Altogether, the antibiofilm potency of monoterpenes

The Journal of Antibiotics Mupirocin and monoterpenes against Staphylococci DKiferet al 694

1,8-cineole 120

100

80

60

Viability (%) 40

* * * * * 20 * 0 ATCC 29213 MFBF 10674 MFBF 10676 MFBF 10677 MFBF 10679 MFBF 10680 S. aureus strains

1,8-cineole (128 mg mL-1) 1,8-cineole (128 mg mL-1) + mupirocin (0.125 µg mL-1)

Figure 4 Biofilm-eliminating efficacy of single 1,8-cineole in comparison with 1,8-cineole-mupirocin combination. Data are represented as viability mean ± s.d. * represent statistically significant difference (Po0.05).

Table 3 Biofilm inhibitory effect of mupirocin–monoterpene combinations

− 1 − 1 S. aureus strain MBICmupirocin (μgml )MBICmonoterpene (μgml )FICIEffect

Thymol ATCC 29213 0.020 0.161 0.43 Synergistic MFBF 10674 0.016 0.131 0.51 Additive MFBF 10676 0.020 0.160 0.45 Synergistic MFBF 10677 0.017 0.137 0.45 Synergistic MRSA 10679 0.021 0.172 0.36 Synergistic MRSA 10680 0.017 0.132 0.45 Synergistic

Menthol ATCC 29213 0.006 0.372 0.60 Additive MFBF 10674 0.003 0.207 0.13 Synergistic MFBF 10676 0.008 0.515 0.35 Synergistic MFBF 10677 0.006 0.383 0.47 Synergistic MRSA 10679 0.008 0.519 0.34 Synergistic MRSA 10680 0.007 0.431 0.38 Synergistic

1.8-cineole ATCC 29213 0.031 15.708 1.01 Indifferent MFBF 10674 0.035 17.718 1.17 Indifferent MFBF 10676 0.032 16.459 0.52 Additive MFBF 10677 0.034 17.390 1.44 Indifferent MRSA 10679 0.043 22.069 1.18 Indifferent MRSA 10680 0.035 17.819 0.89 Additive

Abbreviations: FICI, Fractional Inhibitory Concentration Index; MBIC, minimum biofilm inhibitory concentration.

(thymol4menthol41,8, cineole) against S. aureus presented in this In addition, menthol but not 1,8 cineole was able to increase the study corresponds to available data on antibiofilm efficacy of these eucariotic membrane depolarization, which could be the reason compounds against other Gram- positive and/or Gram-negative behind the changes of ion flux across the membrane, the entry of bacteria.24,33,36–39 The relative hydrophilicity of monoterpenes may Ca2+and loss of Cl − from cytoplasm.42 Monoterpenes applied in facilitate their penetration through the biofilm exopolysaccharide sub-inhibitory concentrations prior to biofilm formation could matrix, whereas their lipophilic nature enables interaction with interact with staphylococcal surface proteins compromising initial bacterial membrane structures.39 Differences in their antibiofilm attachment phase to polystyrene microtitre plates as well as interfering potency may be explained by their octanol-water partition coefficient, with the QS system.12,33,43 40 Po/w. It has been suggested that compounds with a log Po/w value Considering the suggested mechanism of terpenoides antibiofilm higher than 3, such as thymol and menthol, are highly potent in action, we expected to observe potentiated effects of mupirocin– disturbing a cell membrane, while 1,8 cineole with log Po/w 2.8 would monoterpene combinations against mature staphylococcal biofilm. exert weaker effect toward bacterial membrane structures.40,41 Indeed, 1,8-cineol in combination with mupirocin showed a

The Journal of Antibiotics Mupirocin and monoterpenes against Staphylococci DKiferet al 695 potentiated biofilm-eliminating effect, whereas mupirocin–menthol mixtures exerted clear antagonism. We can only speculate that the 1Reiß,S.et al. Global analysis of the Staphylococcus aureus response to mupirocin. simultaneous application of mupirocin and menthol resulted in the Antimicrob. Agents Chemother. 56,787–804 (2012). 2 Brook, I. The role of bacteria in chronic rhinosinusitis. Otolaryngologic Clinics of North formation of some kind of complex with a exopolysaccharide matrix America 38,1171–1192 (2005). that could not disturb a mature biofilm significantly. For thymol– 3 Lin, A. & Busaba, N. Y. Staphylococcus aureus and endoscopic sinus surgery. Curr. fi Opin. Otolaryngol. Head Neck Surg. 14,19–22 (2006). mupirocin combination bio lm eradication potency was inconclusive; 4 Kilty, S. J. & Desrosiers, M. Y. The role of bacterial biofilms and the pathophysiology of the combination exerted potentiated biofilm-eliminating effect against chronic rhinosinusitis. Curr. Allergy Asthma Rep. 8,227–233 (2008). three bacterial strains, to one strain the effect was indifferent and 5Hall-Stoodley,L.et al. Bacterial biofilms: from the natural environment to infectious fi diseases. Nat. Rev. Microbiol. 2,95–108 (2004). against two strains it was antagonistic. Opposite to the bio lm- 6Lewis,K.Riddleofbiofilm resistance. Antimicrob. Agents Chemother. 45,999–1007 eliminating effect, mupirocin–menthol as well as mupirocin–thymol (2001). showed dominant synergism in the inhibition of biofilm formation, 7 Fux, C. A., Stoodley, P., Hall-Stoodley, L. & Costerton, J. W. Bacterial biofilms: a diagnostic and therapeutic challenge. Expert Rev. Anti. Infect. Ther. 1,667–683 whereas mupirocin-1,8 cineole combination exerted an indifferent or (2003). additive effect depending on the S. aureus strain. 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Microbiol (2005) Chapter 1: Unit 1B.1. 30 O’Toole, G. A. Microtiter dish biofilm formation assay. J. Vis. Exp. 47, 2437 (2011). 31 Ceri, H. et al. The Calgary Biofilm Device : new technology for rapid determination of ACKNOWLEDGEMENTS antibiotic susceptibilities of bacterial biofilms the calgary biofilm device : new The authors are grateful to Pliva (Zagreb, Croatia) for providing the mupirocin. technology for rapid determination of antibiotic susceptibilities of bacterial biofilms. fi J. Clin. Microbiol. 37,1771(1999). This work was nancially supported by the University of Zagreb (Grant 32 Ha, K. R., Psaltis, A. J., Butcher, A. R., Wormald, P.-J. & Tan, L. W. In vitro activity of No.1126) and Ministry of Science, Education and Sports of the Republic of mupirocin on clinical isolates of Staphylococcus aureus and its potential implications in Croatia (Grant No. 006–0061117–1242). chronic rhinosinusitis. Laryngoscope 118,535–540 (2008).

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